STRUCTURAL REQUIREMENTS OF PHOSPHOLIPASE-C DELTA-1 FOR REGULATION BY SPERMINE, SPHINGOSINE AND SPHINGOMYELIN

We studied the relationship between sphingomyelin, calcium, spermine a nd sphingosine in regulation of phospholipase C (PLC) delta 1 activity . Inhibition of PLC delta 1 by sphingomyelin was promoted by spermine and Ca2+ and was partially abolished by sphingosine. The effect of sph ingosine and spermine entirely depended on Ca2+. In the absence of Ca2 +, no effect of these substances on PLC delta 1 activity was observed. Using deletion mutants and active fragments of PLC delta 1 generated by limited proteolysis, we have studied the structural requirements of the enzyme for regulation by these compounds. The deletion mutant of PLC delta 1 lacking the first 58 amino acids and the mutant lacking th e entire pleckstrin homology (PH) domain were fully active in the dete rgent assay, and their activities were affected by spermine, sphingosi ne, Ca2+ and sphingomyelin to the same extent as the native enzyme. Th e limited proteolysis of PLC delta 1 generated two fragments of 40 kDa and 30 kDa, which formed a stable active complex. The relationship be tween Ca2+ concentration and enzymatic activity was almost identical f or the native PLC delta 1 and the proteolytic complex. The activity of the proteolytic complex formed by the 40 kDa and 30 kDa peptides was not affected by spermine and sphingosine. Sphingomyelin inhibited the complex slightly less than the native PLC delta 1, and this inhibition was not promoted by spermine. These observations suggest that for act ivation of PLC delta 1 by spermine and sphingosine, the region spannin g domains of high conservation, named X and Y, must be intact. In cont rast, the PH domain and the intact spanning region of the X and Y doma ins are not essential for inhibition of PLC delta 1 by sphingomyelin.